EP0155420B1 - Procédé de préparation d'un corps fongique et d'un lipide riche en acide gamma-linolénique - Google Patents

Procédé de préparation d'un corps fongique et d'un lipide riche en acide gamma-linolénique Download PDF

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Publication number
EP0155420B1
EP0155420B1 EP84306511A EP84306511A EP0155420B1 EP 0155420 B1 EP0155420 B1 EP 0155420B1 EP 84306511 A EP84306511 A EP 84306511A EP 84306511 A EP84306511 A EP 84306511A EP 0155420 B1 EP0155420 B1 EP 0155420B1
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EP
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Prior art keywords
lipid
culture medium
liter
fungal
fungal body
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
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EP84306511A
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German (de)
English (en)
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EP0155420A1 (fr
Inventor
Osamu Suzuki
Toshihiro Yokochi
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National Institute of Advanced Industrial Science and Technology AIST
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Agency of Industrial Science and Technology
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Priority claimed from JP59022394A external-priority patent/JPS60168391A/ja
Priority claimed from JP59115162A external-priority patent/JPS60259192A/ja
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Publication of EP0155420A1 publication Critical patent/EP0155420A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi

Definitions

  • the present invention relates to a method for the preparation of a fungal body and a lipid rich in y-linolenic acid therefrom. More particularly, the invention relates to a method for the preparation of a fungal body of certain filamentous fungi belonging to the genus of Mortierella containing a large amount of lipids by cultivation under specific conditions and to the preparation of a lipid therefrom which is rich in its content of y-linolenic acid.
  • lipids including neutral lipids, i.e. oils and fats, and polar lipids, i.e. phosphilipids and glycolipids, in a content of, for example, 25 to 65% by weight on a dry basis.
  • Such high-lipid filamentous fungus moulds are exemplified by Geotricum candidum, Fusarium lini, Fusarium buligenum, Penicillium lilacinum, Penicillium soppi, Penicillium spinulosum, Aspergillus nidulance and Mucor sacineleudes reported by K.
  • These fungi can be cultured in a culture medium containing a carbohydrate as the carbon source and the preferred concentration of the carbohydrate as the nutrient in the culture medium is reportedly 20 to 60 g/liter when they are cultured in a flask or in a small-size culture tank.
  • the velocity and extent of multiplication of the filamentous fungal body are usually not so high that the carbohydrate as the carbon source is not exhaustively consumed. For example, it is reported by C. G. C. Chester et al (loc.
  • Microbiol., volume 7 (1961), page 895] are that the amount of dried fungal body was 22 g/liter corresponding to only 2.5 g/liter of the lipid extractable therefrom after 6 days of culturing at 30°C starting with a molasses concentration of 164 g/liter (of which about 40% is consumed by the multiplication of the fungus).
  • y-linolenic acid i.e. cis-6,9,12-octadecatrienoic acid which cannot be synthesized in the living body, is one of the essential fatty acids indispensable in the diet for mammals including human beings.
  • y-linolenic acid absorbed by the living body is precursor of and converted into bis-homo-y-linolenic acid and further into arachidonic acid which in turn are converted into prostaglandins E, and F 1 ⁇ and prostaglandins E 2 and F 2a' respectively, playing very important roles in the living body, so that a deficiency of y-linolenic acid in the diet causes various diseases and disorders of the body often requiring the administration of medicines.
  • y-Linolenic acid or a lipic containing the same is conventionally obtained by extraction from the seeds of several plants such as evening primrose (Oenothera biennis L.) but the production thereof is quite unsatisfactory. Therefore, great efforts have been continuously directed, for example, by R. B. Wolt et al. to seek a plant giving seeds containing an oil rich in its content of y-linolenic acid [J. Amer. Oil Chem. Soc., volume 60 (1983), page 1858].
  • the present invention provides a method for the preparation of a fungal body containing a large amount of a lipid rich in its content of y-linolenic acid which comprises culturing a fungal stock selected from Mortierella isabellina, Mortierella vinacea, Mortierella ramanniana, Mortierella ramanniana var.
  • the lipid rich in its content of y-linolenic acid can readily be prepared by extraction from the fungal body, optionally, after separation from the liquid culture medium, for example, using an organic solvent.
  • y-Linolenic acid can be prepared therefrom by concentration according to known techniques.
  • the above described method is based upon the discovery that, when cultured under specific culture conditions, filamentous fungus stocks of Mortierella isabellina, M. vinacea, M. rammanniana, M. ramanniana var. angulispora and M. nana can give fungal bodies having a very high lipid content of from 35 to 70% by weight with high productivity and that the lipid is rich in its content of y-linolenic acid.
  • the possibility of such a high-density culture of Mortierella fungi is quite unexpected because it is generally accepted that filamentous fungi multiplying with hyphae cannot be cultured in a high density condition of the fungal body, in contrast to bacteria and yeast.
  • the scope of the method of the invention for the preparation of a fungal body containing a large amount of lipids consists in the culture of a specific filamentous fungal stock under specific culture conditions.
  • the fungal stocks used in the method of the invention all belong to the genus of Mortierella and kept and available at the Institute of Fermentation, Osaka and also registered in the IFO Catalogue bearing the respective IFO numbers including M. isabellina (IFO 7824, 7873, 7884, 8183, 8308 and 8309), M. vinacea (IFO 6738), M. ramanniana (IFO 8287), M. rammannian var angulispora (IFO 6744 and 8187) and M. nana (IFO 8794).
  • M. isabellina IFO 7824, 7873, 7884, 8183, 8308 and 8309
  • M. vinacea IFO 6738
  • M. ramanniana IFO 8287
  • the medium should contain a carbohydrate as the carbon source which may be, for example, glucose, fructose, saccharose, molasses, starch or the saccharification liquid of wood.
  • the concentration of the carbohydrate in the culture medium should preferably be in the range of from 60 to 400 g per liter or. more preferably, from 80 to 400 g/liter of the medium.
  • the nitrogen source to be added to the culture medium may be an inorganic nitrogen compound such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium phosphate or an organic material such as urea, peptone, yeast extract or corn steep liquor.
  • the culture medium should also contain small amounts of inorganic metal salts such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate, sodium chloride, iron (II) sulfate, magnesium sulfate or zinc sulfate. It is of course optional that certain micronutrients and other nutritive substances may be added to the culture medium, according to need.
  • inorganic metal salts such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate, sodium chloride, iron (II) sulfate, magnesium sulfate or zinc sulfate. It is of course optional that certain micronutrients and other nutritive substances may be added to the culture medium, according to need.
  • the culturing of the above mentioned filamentous fungus is usually performed in a liquid medium with agitation by aeration.
  • the pH value of the culture medium should preferably be in the range of from 4.0 to 6.0 and culturing is continued for 2 to 15 days at a temperature in the range of from 15 to 40°C under agitation at a relatively high velocity of 300 to 800 r.p.m. with aeration at a rate of 0.5 to 2 v.v.m.
  • a fungal body of very high lipid content is produced.in the culture medium in a high density so that the body is separated from the culture medium and the lipid contained therein is extracted by a suitable means such as extraction using an organic solvent.
  • the multiplication of the filamentous fungus takes place with relatively suppressed extension of the hyphae in extremely minute discrete units each containing only 1 to 10 cells, so that the separation of the fungal body from the culture medium is greatly facilitated by use of, for example, a dehydrator or the like machine to give a body with an advantageously low water content of as low as about 60%.
  • the recovery of the lipid from the thus dehydrated fungal body is carried out by a conventional procedure such as by extraction with an organic solvent.
  • the thus obtained lipid is rich in its content of y-linolenic acid which can be separated in a concentrated form from the mixed fatty acids or mixed fatty acid esters by a known method such as the urea adduct process and cooling separation process.
  • the preferred concentration of the acetic acid or acetate in the culture medium is in the range of from 0.1 to 20 g/liter depending on the culturing conditions such as the concentration of the carbon source and others.
  • the pH of the culture medium is preferably in the range from 3.0 to 6.0 and culturing is complete within 10 days.
  • the advantage of the addition of acetic acid or acetate is of course obtained even when the concentration of the carbon source in the culture medium is relatively low such as 20 g/liter but is more marked when the concentration thereof is 60 g/liter or higher.
  • the method of the invention performed in the above described manner can afford the possibility of the production of a lipid with very high productivity because the culturing of the filamentous fungus can be performed in very high densities to give a fungal body of very high lipid content by using a culture medium containing a carbohydrate as the carbon source in a high concentration hitherto not used in the culture of filamentious fungi, together with the ease and high efficiency of the recovery of the lipid from the thus cultured fungal body.
  • the advantage in the productivity is particularly apparent from the calculation of the capacity of the apparatus required for unit production of the lipid.
  • the hourly production of the fungal body per unit volume of the culture medium would be 1.7 g fungal body per liter per hour assuming a fungal body multiplication of 100 g/liter medium and in consideration of the culture time necessary to obtain this multiplication estimated from the results of the experimentation described below. Therefore, a constant yearly running of 1000 m 3 of the culture medium, which is in practice possible by a combined cycling use of 3 culture tanks each having a capacity of 800 m 3 , would provide a yearly fungal body production of 14,000 tons/year corresponding to a yearly lipid production of 7,000 tons/year assuming a 50% content of the lipid in the body.
  • the lipid produced in this manner contains more than 95% of fats or oils, i.e. triglycerides, so that it is useful not only as a food but also as a starting material for the manufacture of various products derived from fats and oils.
  • the phospholipids and glycolipids contained in the lipid are useful as ingredients of medicines or as surface active agents.
  • the fungal body residue after extraction of the lipid is composed mainly of proteins and nucleic acids which are useful, respectively, as a provender and medicines.
  • the present invention provides an industrial process for microbiologically producing a lipid containing a large amount of unsaturated fatty acids or, in partiuclar, y-linolenic acid with high productivity.
  • the method of the invention is very useful as a method for the preparation of a y-linolenic acid-containing lipid or as a method for the modification of a lipid.
  • Particular advantages are obtained in the method of preparation of a y-linolenic acid-containing lipid as a substitute for conventional methods starting with the extraction of seeds of specific plants.
  • a culture medium was prepared by dissolving 30 g of glucose, 2 g of potassium dihydrogenphosphate KHZPO4, 0.3 g of magnesium sulfate MgSO 4 ⁇ 7H 2 O, 0.1 g of sodium chloride NaCI, 10 mg of iron (II) sulfate FeS04. 7H 2 0, 10 mg of calcium chloride CaCI2. 2H 2 0, 0.2 mg of copper sulfate CuSO 4 ⁇ 5H 2 O, 1.0 mg of zinc sulfate ZnS0 4 - 7H 2 0, 1.0 mg of manganese chloride MnCI 2 .
  • a 400 ml portion of the thus prepared culture medium taken in an Erlenmeyer flask of 1000 ml capacity was inoculated with one of the Mortierella fungi named before and culture of the fungus was performed by shaking the flask at a velocity of 150 r.p.m. for 7 days at a culture temperature of 30°C.
  • the fungal body in the medium was collected by filtration or centrifugal separation.
  • a portion of the collected fungal body was used for the determination of the water content therein by exactly weighing before and after drying by heating for 24 hours in an air oven at 120°C. The remainder portion of the wet fungal body was subjected to the extraction of the lipid.
  • the wet fungal body obtained by filtration was added to a 2:1 by volume mixture of chloroform and methyl alcohol and homogenized in the presence of glass beads to effect simultaneous grinding of the body and extraction of the lipid from the ground body into the solvent.
  • This extraction procedure was repeated 5 times each with a fresh portion of the solvent and all the extract solutions were combined together.
  • the solvents were removed from the combined extract solution by evaporation under reduced pressure and the amount of the total lipid was determined by weighing the residual matter.
  • the thus extracted lipid was converted to the methyl ester according to a conventional procedure and the mixture of the methyl esters was analyzed by gas chromatography to determine the relative fatty acid composition.
  • Table 1 gives the results of the culture tests comparing the cultures with or without the addition of acetic acid or acetate together with the yields of the fungal body and lipid each in g per liter of the medium, the content of the lipid in % in the fungal body, the fungal body coefficient, i.e. yield of the fungal body in g per 100 g of glucose consumption, the lipid coefficient, i.e. yield of the lipid in g per 100 g of glucose consumption, the content of y-linolenic acid in % in the overall fatty acids, the content of unsaturated fatty acids in % in the overall fatty acids and the unsaturation factor, i.e. the average number of unsaturated linkages per molecule of the overall fatty acids.
  • the addition of acetic acid or acetate to the culture medium always increases the yield of the fungal body for the same species of fungus reaching twice or more in most of the species in comparison with the culture without the addition thereof.
  • This advantageous effect is even more remarkable when a comparison is made of the yield of the lipid which increases 1.5 to 6 times with the addition of acetic acid or acetate.
  • the content of the lipid in the fungal body was increased by 10 to 20% by the addition of acetic acid or acetate in some cases. Accordingly, the fungal body coefficient and lipid coefficent, i.e. the yields of the fungal body and lipid in g per 100 g consumption of glucose, respectively, were greatly increased by the addition of acetic acid or acetate.
  • the content of y-linolenic acid was always increased by the addition of acetic acid or acetate and an increase of 1.7 to 20.1 % was noted in the content of the unsaturated fatty acids.
  • the addition of acetic acid or acetate had the effect not only of increasing the overall yield of the unsaturated fatty acids, but also increasing the degree of unsaturation, i.e. the average number of unsaturated linkages per molecule, in the unsaturated fatty acids.
  • the results of this example show that the nature of the lipid can be improved or modified in such a way that the yield of the unsaturated fatty acids including y-linolenic acid as a highly unsaturated fatty acid is increased by the addition of acetic acid or acetate to the culture medium.
  • Culture media were prepared each by dissolving in 1000 ml of deionized water an amount of from 85 to 200 g/litre of glucose as the carbon source, 2 g of potassium dihydrogenphosphate KH z P0 4 , 0.3 g of magnesium sulfate MgS04. 7H 2 0, 0.1 g of sodium chloride NaCI, 0.2 g of malt extract, 0.2 g of yeast extract, 0.1 g of peptone, 10 mg of iron (II) sulfate FeS04. 7H 2 0, 19 mg of calcium chloride CaC12. 2H 2 0, 0.2 mg of copper (Il) sulfate C U S0 4 .
  • a culture tank of 10 liters or 30 liters capacity were introduced 6 liters or 20 liters, respectively, of the above prepared culture medium into which Mortierella ramanniana var. angulispora IFO 8187 was inoculated and cultured therein at 30°C or 35°C under agitation at a velocity of 300 to 700 r.p.m. with aeration at a rate of 0.5 to 2.0 v.v.m.
  • a 100 ml portion of the culture medium was periodically taken out of the tank and filtered to separate the fungal body from the culture medium.
  • a part of the thus collected body was subjected to the determination of the water content by drying in a thermostat at 120°C for 24 hours after exact weighing and the remainder was used for the extraction of the lipid.
  • a varied amount of sodium acetate indicated in Table 2 was further added to the culture medium to examine the effect of acetate addition.
  • Cultures No. 21 to No. 29 were each performed in a culture tank of 10 liter capacity and Cultures No. 30 to No. 32 were each performed in a culture tank of 30 liter capacity.
  • the addition of sodium acetate had the effect of slightly decreasing the yield of the fungal body while significant improvement was obtained in the content of the lipid in the fungal body by 10% or more corresponding to an increase in the yield of the lipid by 20% or more and accordingly to a remarkable improvement in the lipid coefficient indicating that the effect of sodium acetate is not critically dependent on the concentration thereof at least in the range from 0.3 to 5 g/liter.

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Claims (8)

1. Procédé de préparation d'un lipide riche en teneur en acide y-linolénique qui comprend:
(a) la mise en culture d'un champignon d'une espèce appartenant au genre de Mortierella choisi dans le groupe consistant en M. isabellina, M. vinacea, M. ramanniana, M. ramanniana var. angulispora et M. nana dans un milieu liquide de culture contenant un carbohydrate en tant que source de carbone à une concentration de 20 à 400 g/liter pour faire croître le corps fongique du champignon contenant un lipide, ledit milieu liquide de culture contenant de l'acide acétique ou un acétate d'un métal alcalin ou de l'acétate d'ammonium à une concentration de 0,1 à 20 g/litre; et
(b) l'extraction du lipide du corps fongique.
2. Procédé selon la revendications 1 où le carbohydrate est du glucose ou de la mélasse.
3. Procédé selon la revendication 1 ou la revendication 2 où la concentration du carbohydrate dans le milieu liquide de culture est comprise entre 60 et 400 g/litre.
4. Procédé selon l'une quelconque des revendications 1 à 3 où l'acétate d'un métal alcalin est l'acétate de sodium ou de potassium.
5. Procédé selon l'une quelconque des revendications précédentes où la valeur de pH du milieu liquide de culture est comprise entre 3,0 et 6,0.
6. Procédé selon l'une quelconque des revendications précédentes où une source d'azote est ajoutée au milieu de culture.
7. Procédé selon l'une quelconque des revendications précédentes où la mise en culture est continuée pendwnt 2 à 15 jours à une température comprise entre 15 et 40°C avec agitation à une vitesse de 300 à 800 t/mn et aération à raison de 0,5 à 2,0 v.v.m.
8. Procédé selon l'une quelconque des revendications précédentes où le corps fongique est séparé du milieu de culture et le lipide est alors extrait du corps fongique par un solvant organique.
EP84306511A 1984-02-09 1984-09-25 Procédé de préparation d'un corps fongique et d'un lipide riche en acide gamma-linolénique Expired EP0155420B1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP59022394A JPS60168391A (ja) 1984-02-09 1984-02-09 γ‐リノレン酸濃縮物の製造方法
JP22394/84 1984-02-09
JP115162/84 1984-06-05
JP59115162A JPS60259192A (ja) 1984-06-05 1984-06-05 微生物脂質の生産方法

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EP0155420A1 EP0155420A1 (fr) 1985-09-25
EP0155420B1 true EP0155420B1 (fr) 1988-03-23

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US (1) US4783408A (fr)
EP (1) EP0155420B1 (fr)
CA (1) CA1235083A (fr)
DE (1) DE3470061D1 (fr)

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JPS6137096A (ja) * 1984-07-31 1986-02-21 Nisshin Oil Mills Ltd:The γ−リノレン酸含量の高い脂質成分の製造法
US4870011A (en) * 1985-01-22 1989-09-26 Director General Of Agency Of Industrial Science And Technology Method for obtaining lipids from fungus bodies
JPH0716424B2 (ja) * 1985-10-01 1995-03-01 ライオン株式会社 アラキドン酸含有脂質の製造方法
US5204250A (en) * 1986-03-31 1993-04-20 Suntory Limited Process for production of arachidonic acid
CA1317901C (fr) * 1986-07-08 1993-05-18 Yoshifumi Shinmen Procede d'obtention d'acides bihomo-_-linolenique et eicosapentaenoique
JPH0712314B2 (ja) * 1986-07-08 1995-02-15 サントリー株式会社 γ―リノレン酸及びこれを含有する脂質の製造方法
JPS63133994A (ja) * 1986-11-21 1988-06-06 Lion Corp γ−リノレン酸を含有する脂質の製造方法
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DE3785023T3 (de) * 1987-01-28 1998-12-17 Suntory Limited, Osaka Verfahren zur Herstellung von Arachidonsäure.
US5034321A (en) * 1987-08-19 1991-07-23 Idemitsu Petrochemical Co., Ltd. Method for the production of lipids containing bis-homo-γ-linolenic acid
JPH01228486A (ja) * 1988-03-09 1989-09-12 Suntory Ltd 奇数鎖高度不飽和脂肪酸及びこれを含有する脂質の製造方法
JP2710344B2 (ja) * 1988-07-13 1998-02-10 サントリー株式会社 高度不飽和脂肪酸及びこれを含有する脂質の製造方法
US20060094089A1 (en) * 1988-09-07 2006-05-04 Martek Biosciences Corporation Process for the heterotrophic production of microbial products with high concentrations of omega-3 highly unsaturated fatty acids
US5340742A (en) 1988-09-07 1994-08-23 Omegatech Inc. Process for growing thraustochytrium and schizochytrium using non-chloride salts to produce a microfloral biomass having omega-3-highly unsaturated fatty acids
US5093249A (en) * 1989-05-24 1992-03-03 Idemitsu Petrochemical Co., Ltd. Process for production of dihomo-γ-linolenic acid and inhibitor for unsaturation reaction at Δ5-position of fatty acid
JP3354582B2 (ja) * 1991-09-30 2002-12-09 サントリー株式会社 オメガ９系高度不飽和脂肪酸およびこれを含有する脂質の製造方法
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US5583019A (en) 1995-01-24 1996-12-10 Omegatech Inc. Method for production of arachidonic acid
US6255505B1 (en) 1996-03-28 2001-07-03 Gist-Brocades, B.V. Microbial polyunsaturated fatty acid containing oil from pasteurised biomass
CN1226923B (zh) * 1996-03-28 2011-06-15 Dsmip资产有限公司 粒状微生物生物量的制备及其有价值的化合物的分离方法
CA2250581C (fr) * 1996-03-28 2008-08-12 Gist-Brocades B.V. Preparation d'acide gras polyinsature microbien a partir d'huile contenant une biomasse pasteurisee
JP3792309B2 (ja) 1996-08-30 2006-07-05 サントリー株式会社 不飽和脂肪酸含有油脂の製造方法
ES2446982T3 (es) * 1996-12-27 2014-03-11 Suntory Holdings Limited Medios para cultivar microorganismos y método para producir ácidos grasos insaturados o lípidos que los contienen
EP2341127B1 (fr) 2000-01-28 2015-05-27 DSM IP Assets B.V. Production amelioree de lipides contenant des acides gras polyenes au moyen de cultures a grande densite de microbes eucaryotes dans des fermenteurs
JP4088097B2 (ja) * 2002-04-26 2008-05-21 サントリー株式会社 高度不飽和脂肪酸含有脂質の製造方法
DE60325590D1 (de) 2002-06-19 2009-02-12 Dsm Ip Assets Bv Pasteurisationsverfahren für mikrobielle zellen und mikrobielles öl
US20110177564A1 (en) * 2010-01-15 2011-07-21 Massachusetts Institute Of Technology Bioprocess and microbe engineering for total carbon utilization in biofuel production

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US4783408A (en) 1988-11-08
CA1235083A (fr) 1988-04-12
EP0155420A1 (fr) 1985-09-25

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